In this study, we present a facile and scalable preparation of formamide-functionalized and oxygen-deficient V2O5 nanosheets (thickness: 30 ∼ 40 nm) modified electrode (FTO|exf-V2O5) with promising peroxidase-like and photoelectrocatalytic activity. FTO|exf-V2O5 was prepared by the direct top-down exfoliation of the V2O5 sub-micron plates (thickness: 300 ∼ 550 nm) modified electrode (FTO|micro-V2O5) in the presence of formamide as the exfoliation agent at mild conditions. Formamide molecules were found to adsorb on FTO|exf-V2O5 and serve as the active sites for the reduction of noble metal, which not only enables the application of FTO|exf-V2O5 for the recovery of noble metals but also allows further functionalization and electrocatalytic oxidation of H2O2. As compared with FTO|micro-V2O5, FTO|exf-V2O5 had significantly higher oxygen vacancy (VO) content and exhibited remarkably enhanced peroxidase activity with an ultra-low Michaelis-Menten constant of 68.24 μM and a high catalytic constant (or maximal turnover frequency) of 0.28 s−1. Finally, FTO|exf-V2O5 with lower dimension and higher VO content showed improved kinetics of charge-transport and interfacial charge-transfer, resulting in the enhanced activity towards photoelectrocatalytic oxidation of water and methanol. This work opens a new avenue for the preparation of exfoliated 2-D material based platform for biomimetic catalysis and energy conversion.